Methods of cell separation

ABSTRACT

The present invention relates to the use of a combination of:
         (i) a macromolecular erythrocyte sedimentation enhancer, and   (ii) dimethyl sulphoxide (DMSO), dimethylglycine (DMG) and/or valine;
 
to recover non-erythrocyte blood cells from a blood cell-containing sample and/or to prime non-erythrocyte blood cells to protect their integrity in subsequent cryopreservation step(s).

This invention relates to methods and compositions for separating and/orpriming cells, and more particularly to methods and compositions forseparating erythrocytes from peripheral blood, bone marrow, umbilicalcord blood and related blood tissues.

Isolation of white cells from blood for in vitro studies or in cellulartherapy usually incorporates an initial separation of blood mainly basedon the bulk depletion of erythrocytes, which comprise >99% of thecellular mass.

Techniques for erythrocyte removal are based on hypotonic lysis oferythrocytes, density gradient separation, enhanced centrifugalsedimentation using hydroxyethyl starch or mixtures (containing amongstother things antibodies) that promote accelerated sedimentation of theerythrocytes.

Hypotonic lysis, while useful in low volume in vitro studies, can beimpractical for the large volumes of blood tissues processed forcellular therapies. In cell therapy procedures, erythrocyte hypotoniclysis is usually performed as a final step to remove the remainingcontaminating erythrocytes in a sample after bulk depletions by othermethods.

Density-gradient separation relies on differences in the densities ofcell types that causes them to segregate at different levels in a fluidmedium of variable density. Differences in density between the celltypes can be small, and different cell types can be heterogeneous insize and density. Consequently, particular cell types can becomedistributed throughout a density-gradient medium rather than preciselysegregating at a discrete area in the density medium, resulting inreduced recovery of desired cells and/or contamination with undesiredcell types.

In procedures that enrich for rare blood cell types such ashematopoietic progenitor cells, density-gradient sedimentation can leadto loss or reduced yields of desired cell subsets. For example, usingconventional density-gradient methods to isolate progenitor cells fromumbilical cord blood results in a significant loss of the desired stemcells e.g., CD34⁺ hematopoietic stem cells (HSCs) or VSELs (Very SmallEmbryonic Like Stem Cells) (Wagner, J. E., Am J Ped Hematol Oncol 15:169(1993)). Using conventional density-gradient methods to isolatelymphocytes can result in selective loss of particular lymphocytesubsets. (Collins, J Immunol Methods 243:125(2000)).

These separation methods have an additional contraindication for use incellular therapies in that the chemical entities in the separationmedium can be toxic if infused with the cells into the recipient. Assuch, additional steps must be performed to ensure their completeremoval prior to infusion. Instrument methodologies such as elutriationalso depend upon differential separation of blood components by densityand can suffer from similar deficiencies in performance.

Another method for removing erythrocytes from blood utiliseshydroxyethyl starch which stimulates erythrocyte aggregation, which thensediment more rapidly than leukocyte components when sedimented at 50×gin a centrifuge. While this method is generally non-toxic for therecipient, its performance in the recovery of important cell types,including, for example, mesenchymal stem cells (MSCs) hematopoietic stemcells (HSCs) and VSELs is variable and with respect to umbilical cordblood, for example, can result in less-than-ideal recovery of stem cellsand diminution of the engraftment potential of the cord blood cells,increasing the risk for transplant failure.

A more recently reported methodology has been disclosed (EP 2,117,592;U.S. Pat. No. 7,598,089) and involves the mixing of uncoaggulated bloodwith a composition comprising: dextran; anti-glycophorin A antibody; inaddition to other antibody species (e.g. anti-CD9 antibodies; anti-CD15antibody; and tandem antibodies, in which two different antibodies havebeen joined together to form a single entity). Antibodies represent anexpensive solution to methods of cell separation and recovery ofnucleated cells of interest from blood.

Increasing the recovery of rare cell types from donor tissue woulddramatically improve the outcome of transplant and immune therapies(e.g., bone marrow transplants, stem cell-based gene therapy, and immunecell therapy), the success of which is related to the actual number ofthe cells being introduced for the therapeutic application. It is alsodesirable to provide a method of cell separation which results in highproportions of viable cells of interest.

SUMMARY OF THE INVENTION

The present invention relates to the finding that certain compounds,namely dimethyl sulphoxide (DMSO) and certain amino acids, havesurprisingly advantageous effects when brought into contact with bloodcells in combination with a macromolecular erythrocyte sedimentationenhancer—some of these effects become apparent only when the blood cellsat issue are subsequently subjected to certain processing. Inparticular, the use of these compounds in combination with amacromolecular erythrocyte sedimentation enhancer enables the recoveryof higher quantities of non-erythrocyte blood cells from a bloodcell-containing sample. These compounds can also be used in combinationwith a macromolecular erythrocyte sedimentation enhancer to primenon-erythrocyte blood cells so as to protect their integrity insubsequent cryopreservation step(s), thus also enabling higherquantities of non-erythrocyte blood cells to be recovered followingcryopreservation. Thus, the present inventor has developed a method oftreating blood samples to achieve separation of red and white bloodcells. The method also accelerates the separation of red and white bloodcell fractions and does not rely on the use of antibodies. The presentinventor has also developed a method of priming a cell fraction forcryopreservation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides the use of a combination of:

(i) a macromolecular erythrocyte sedimentation enhancer, and

(ii) dimethyl sulphoxide (DMSO), dimethylglycine (DMG) and/or valine;

to recover non-erythrocyte blood cells from a blood cell-containingsample and/or to prime non-erythrocyte blood cells to protect theirintegrity in subsequent cryopreservation step(s). Preferably, themacromolecular erythrocyte sedimentation enhancer is dextran andcomponent (ii) is DMSO.

When the combination of components (i) and (ii) is used to recovernon-erythrocyte blood cells from a blood cell-containing sample,component (ii) can be used to enable the recovery of more viablenon-erythrocyte blood cells from the blood cell-containing sample thanwould be obtained using component (i) alone.

When components (i) and (ii) are used in accordance with the presentinvention, the components are typically used at relatively lowconcentrations, compared to e.g. the concentrations at which agents suchas dextran and/or DMSO may be used when employed as cryoprotectants.When the non-erythrocyte blood cells are contacted with components (i)and (ii), the resulting mixture may comprise component (i) at aconcentration of 0.01 to 20% w/v, preferably 0.05 to 10% w/v, e.g. 0.1to 5% w/v (such as 0.5 to 2% w/v, 1 to 1.5% w/v, or at or around 1.25%w/v), or 2 to 10% w/v (such as 5 to 7.5% w/v, 6 to 6.5% w/v, or at oraround 6.25% w/v). Preferably, when the non-erythrocyte blood cells arecontacted with components (i) and (ii), the mixture comprises component(ii) at a concentration of 0.01 to 20% v/v, preferably 0.05 to 10% v/v,e.g. 0.1 to 5% v/v (such as 0.5 to 2% v/v, 1 to 1.5% v/v, or at oraround 1.25% v/v), or 2 to 10% v/v (such as 5 to 7.5% v/v, 6 to 6.5%v/v, or at or around 6.25% v/v). Typically, the concentration ofcomponent (i) in % w/v is approximately the same as the concentration ofcomponent (i) in % v/v, although the concentration of component (ii) mayat times be higher, e.g. up to two, three, four, or five times higher.

Preferably, when the combination of components (i) and (ii) is used toprime non-erythrocyte blood cells to protect their integrity insubsequent cryopreservation step(s), this priming step is distinct fromany steps that would be taken in order to place the cells in a formready for direct cryopreservation/freezing. In other words, before theprimed non-erythrocyte blood cells are subjected to cryopreservation, afurther step of adding an appropriate amount of cryoprotectant isappropriate. In this regard, for the formulation intended forcryopreservation it may be advantageous to use a cryoprotectant whichcomprises at least one of components (i) or (ii), which will be presentalready from the priming step. That way, there is no need to recover thecells from the priming composition—rather, one or more additionalcryoprotectant agents can just be added to the mixture of the cells andthe priming composition.

Preferably, when the combination of components (i) and (ii) is used toprime non-erythrocyte blood cells to protect their integrity insubsequent cryopreservation step(s), the subsequent cryproservationsstep(s) may refer to one or more of the steps of (b) adding acryoprotectant to the thus obtained non-erythrocyte blood cells, (c)cryopreserving the non-erythrocyte blood cells, (d) thawing thenon-erythrocyte blood cells, and/or (e) recovering the non-erythrocyteblood cells from the cryopreserved formulation. Typically it refers tothe step (b), to step (c), or to both of steps (b) and (c), as these aresteps where the combination of components (i) and (ii) is particularlyeffective in helping protect cell integrity against the type of damagethat may otherwise occur in the absence of the combination of components(i) and (ii) (i.e. even if just one of these components was present inthe absence of the other).

As will be appreciated, components (i) and (ii) can of course be used toprime non-erythrocyte blood cells in the form of a cell fraction thathas already undergone a treatment(s) to remove erythrocytes and/or oneor more specific nucleated cells so as to attain a specific desiredsubset of white blood cells. However, since the cell separation methodof the present also serves to protect the resulting(separated/recovered) cells against damage by subsequentcryopreservation step(s)), simply using the cell separation method ofthe present invention can be much more efficient, as benefits similar tothose which arise in the priming method of the invention can be enjoyedwithout the need for two separate (separation and priming) treatmentsteps.

Thus, the present invention provides a method for separating cells, saidmethod comprising:

-   -   (a) contacting a blood cell-containing sample with:        -   (i) a macromolecular erythrocyte sedimentation enhancer, and        -   (ii) DMSO, DMG and/or valine;    -   (b) allowing said sample to partition into a sedimented phase        and a supernatant phase; and    -   (c) recovering cells from said sedimented and/or supernatant        phase.

The combination of components (i) and (ii) enables the recovery of moreviable non-erythrocyte blood cells than could be obtained usingcomponent (i) alone. This advantage arises both (a) following theseparation step, and (b) following subsequent cryopreservation step(s).

Preferably, step (a) comprises contacting a blood cell-containing samplewith a composition comprising the components (i) and (ii). As mentionedbelow, a preferred agent for use as component (i) is dextran.

Preferably, component (ii) is DMSO, DMG or valine.

Preferably, step (c) comprises recovering cells from said sedimented orsaid supernatant phase.

Thus, in a preferred aspect, the present invention provides a method forseparating cells, said method comprising:

-   -   a) contacting a blood cell-containing sample with a composition        comprising:        -   I) dextran; and        -   II) dimethyl sulphoxide (DMSO), dimethylglycine (DMG) or            valine    -   b) allowing said sample to partition into a sedimented phase and        a supernatant phase; and    -   c) recovering cells from said sedimented phase or said        supernatant phase.

Preferably, in the method of the invention for separating cells, step(a) comprises contacting a blood cell-containing sample with acomposition comprising components (i) and (ii), wherein the blood cellcontaining sample and the composition are mixed at a ratio of 10:1 to1:10 by volume (ratios of components mentioned herein are generallyintended to refer to ratios by volume unless indicated otherwise),preferably 5:1 to 1:5, more preferably 2:1 to 1:2. Typically, the ratiois at or about a ratio of 1:1.

The exact ratio used may depend on the concentration of components (i)and (ii) in the composition. Thus, one particularly preferredcomposition for use in the present invention is a composition that isbased on PBS, and which is prepared by combining equal amounts (byvolume) of 5% w/v Dextran 500 in PBS, and 5% v/v DMSO in PBS (unlessindicated otherwise, concentrations of component (i) mentioned hereinare generally intended to refer to units of % w/v, and concentrations ofcomponent (ii) mentioned herein are generally intended to refer to unitsof % v/v). This composition is referred to herein as “TotiCyte 1×”, andcontains 2.5% w/v Dextran 500 and 2.5% v/v DMSO in PBS. Theabove-mentioned ratios are particularly preferred when this compositionis being used. However, the concentration of components (i) and (ii) inthe composition can be varied. For instance, an alternative compositionfor use according to the invention is a more concentrated formulationreferred to herein as “TotiCyte 5×” refers to a TotiCyte compositionbased on PBS and containing 7.5% w/v Dextran 500 and 7.5% v/v DMSO. Whencompositions such as this one are being used, the preferred ratios maybe adjusted so as to decrease the proportion of the composition, e.g. toa third, a quarter, or fifth. Thus, in this embodiment it may be morepreferred for the blood cell containing sample and the composition to bemixed at a ratio of 50:1 to 1:2 by volume, preferably 25:1 to 1:1, morepreferably 10:1 to 5:2. Typically, the ratio is at or about a ratio of5:1.

Thus, generally in the context of the present invention, it is preferredfor the blood cell containing sample and the composition to be mixed ata ratio of 50:1 to 1:10 by volume, preferably 25:1 to 1:5, morepreferably 10:1 to 1:2.

The ratio at which to mix the blood cell containing sample withcomponents (i) and (ii) may also be defined with reference to theconcentration of components (i) and (ii) in the resulting mixture.Preferably the blood cell-containing sample and component (i) arecombined so as to provide a concentration of component (i) in theresulting mixture of 0.1 to 8% w/v, preferably 0.25 to 5% w/v, morepreferably 0.5 to 2% w/v, yet more preferably 1 to 1.5% w/v, andtypically at or around 1.25% w/v. Preferably the blood cell-containingsample and component (ii) are combined so as to provide a concentrationof component (ii) in the resulting mixture of 0.1 to 8% v/v, preferably0.25 to 5% v/v, more preferably 0.5 to 2% v/v, yet more preferably 1 to1.5% v/v, and typically at or around 1.25% v/v. Typically theconcentrations for components (i) and (ii) are approximately the same.Thus, in one preferred aspect of the separation method of the invention,once the blood cell-containing sample has been contacted with components(i) and (ii), the concentration of component (i) is 0.25 to 5% w/v, andthe concentration of component (ii) is 0.25 to 5% v/v.

Target concentrations in the (cell+composition) mixtures can be higherif a more concentrated composition of components (i) and (ii) is beingused, such as TotiCyte 5×. Thus, more generally the target concentrationfor component (i) is 0.1 to 10% w/v, preferably 0.5 to 5% w/v, morepreferably 1 to 4% w/v, and typically at or around 1.25 or 2% w/v; andthe target concentration for component (ii) is 0.1 to 10% v/v,preferably 0.5 to 5% v/v, more preferably 1 to 4% v/v, and typically ator around 1.25 or 2% v/v.

In the cell separation method of the invention, when step (a) comprisescontacting a blood cell-containing sample with a composition comprisingcomponents (i) and (ii), this composition preferably comprises 0.5-10%(more preferably 1-5%, typically 2-4%) w/v of component (i) and 0.5-10%(more preferably 1-5%, typically 2-4%) v/v of component (ii).

Preferably, the blood cell-containing sample on which the method of theinvention is carried out is selected from peripheral blood, umbilicalcord blood and bone marrow.

Preferably, the blood cell-containing sample on which the method of theinvention is carried out is taken from a human. It is also preferred forthe blood cell-containing sample to contain an anticoagulant. The natureof the anticoagulant is not particularly limited—typical examplesinclude heparin, EDTA, CPD and CPDA, with CPD and CPDA being preferred,and CPDA most preferred.

Preferably, the separation method of the present invention is forrecovering non-erythrocyte blood cells from a blood cell-containingsample containing erythrocyte blood cells. Thus, typically the removessubstantially all of the erythrocytes, e,g, at least 90%, preferably atleast 95%, more preferably at least 98% and typically at least 99% ofthe erythrocytes present in the starting blood cell-containing sample.

Preferably, the separation method of the present invention is for use inpreparing a sample of non-erythrocyte blood cells having a haematocritof less than 1% (by volume). Thus, the cells that are recovered fromsaid sedimented and/or supernatant phase in step (c) preferably have ahaematocrit of no more than 1%, and more preferably less than 1%.

In one embodiment, the separation method of the present invention is forpreparing a sample of concentrated platelets.

The methods of the present invention enable the recovery of enhancedproportions of non-erythrocyte blood cells, typically white blood cells.In the context of the separation method of the invention, the level ofrecovery for the or each of the desired non-erythrocyte blood celltype(s) is preferably at least 80%, preferably at least 90%, and morepreferably at least 95% relative to the amount in the starting wholeblood sample. The post thaw recovery levels obtainable by using theseparation or priming methods of the invention preferably enablerecovery levels of at least 60%, more preferably at least 80%, andtypically at least 90% (particularly if the recovery levels are measuredbefore any washing steps that may be carried out post thaw). Suitablemethods for measuring the concentration of such desired cell types, suchas the total nucleated cell (TNC), CD34 and CD45 cell counts arediscussed below in the Examples.

In the separation method of the invention, in step (b) the sample ispreferably allowed to partition into a sedimented phase and asupernatant phase for 10 to 60 minutes.

The present invention also provides a method for priming anon-erythrocyte blood cell fraction for crypreservation, said methodcomprising contacting the cell fraction with a combination of:

(i) a macromolecular erythrocyte sedimentation enhancer, and

(ii) DMSO, DMG and/or valine;

wherein when the blood cell-containing sample is contacted withcomponents (i) and (ii), the concentration of component (ii) does notexceed 5% v/v. Preferably in this regard, the concentration of component(ii) does not exceed 4% v/v, more preferably it does not exceed 3% v/v,more preferably still it does not exceed 2% v/v, and typically it doesnot exceed 1.5% v/v.

Preferred aspects described above for the use of the components (i) and(ii) in the separation method of the invention are also preferred forthe priming method of the invention.

For instance, the priming method of the invention preferably comprisescontacting the cell fraction with a composition comprising components(i) and (ii), wherein the cell fraction and the composition are mixed ata ratio of 10:1 to 1:10 by volume, preferably 5:1 to 1:5, morepreferably 2:1 to 1:2. Typically, the ratio is at or about a ratio of1:1.

As above, though, the exact ratio used may depend on the concentrationof components (i) and (ii) in the composition. Thus, the above-mentionedratios are particularly preferred when the “TotiCyte 1×” composition ofthe invention is being used. However, the concentration of components(i) and (ii) in the composition can be varied. For instance, when the“TotiCyte 5×” composition of the invention is being used, the preferredratios may be adjusted so as to decrease the proportion of thecomposition, e.g. to a third, a quarter, or a fifth. Thus, in thisembodiment it is more preferred for the cell fraction and thecomposition to be mixed at a ratio of 50:1 to 1:2 by volume, preferably25:1 to 1:1, more preferably 10:1 to 5:2. Typically, the ratio is at orabout a ratio of 5:1.

Thus, generally in the context of the present invention, it is preferredfor the cell fraction and the composition to be mixed at a ratio of 50:1to 1:10 by volume, preferably 25:1 to 1:5, more preferably 10:1 to 1:2.

The ratio at which to mix the cell fraction with components (i) and (ii)may also be defined with reference to the concentration of components(i) and (ii) in the resulting mixture. Preferably the cell fraction andcomponent (i) are combined so as to provide a concentration of component(i) in the resulting mixture of 0.1 to 8% w/v, preferably 0.25 to 5%w/v, more preferably 0.5 to 2% w/v, yet more preferably 1 to 1.5% w/v,and typically at or around 1.25% w/v. Preferably the cell fraction andcomponent (ii) are combined so as to provide a concentration ofcomponent (ii) in the resulting mixture of 0.1 to 8% v/v, preferably0.25 to 5% v/v, more preferably 0.5 to 2% v/v, yet more preferably 1 to1.5% v/v, and typically at or around 1.25% v/v. Typically theconcentrations for components (i) and (ii) are approximately the same.Thus, in one preferred aspect of the priming method of the invention,once the cell fraction has been contacted with components (i) and (ii),the concentration of component (i) is 0.25 to 5% w/v, and theconcentration of component (ii) is 0.25 to 5% v/v.

Target concentrations in the (cell+composition) mixtures can be higherif a more concentrated composition of components (i) and (ii) is beingused, such as TotiCyte 5×. Thus, more generally the target concentrationfor component (i) is 0.1 to 10% w/v, preferably 0.5 to 5% w/v, morepreferably 1 to 4% w/v, and typically at or around 1.25 or 2% w/v; andthe target concentration for component (ii) is 0.1 to 10% v/v,preferably 0.5 to 5% v/v, more preferably 1 to 4% v/v, typically at oraround 1.25 or 2% v/v, and most typically at or around 1.25% v/v.

The priming method of the invention preferably comprises contacting thecell fraction with a composition comprising components (i) and (ii),wherein the composition comprises 0.1-10% (more preferably 0.5-5%,typically 1-2%) w/v of component (i) and 0.1-10% (more preferably0.5-5%, typically 1-2%) v/v of component (ii).

Preferably, the cell fraction comprises white blood cells.

Preferably, the cell fraction is taken from a human.

In a preferred aspect, the separation and priming methods of the presentinvention are for (the purpose of) increasing the proportion of viablewhite blood cells recovered following subsequent cryopreservationstep(s). In particular, introducing component (ii) enhances the quantityof viable cells that can be recovered following cryopreservation, ascompared to what is possible with component (i) alone. As noted above,this reference to cryopreservation step(s) may refer to one or more ofthe steps of (b) adding a cryoprotectant to the thus obtainednon-erythrocyte blood cells, (c) cryopreserving the non-erythrocyteblood cells, (d) thawing the non-erythrocyte blood cells, and/or (e)recovering the non-erythrocyte blood cells from the cryopreservedformulation, but typically it refers to step (b), to step (c), or toboth of steps (b) and (c).

As regards component (i) for use in accordance with the methods andcompositions of the present invention, there is no specific limitationas to the type of macromolecular erythrocyte sedimentation enhancer thatcan be used. Possible substances that may be used include polybrene,protamine sulphate, polyethylene glycol (PEG), hydroxyethyl starch(HES), polyvinyl pyrrolidone (PVP), and dextrans (preferably dextranswith a molecular weight of at least around 50 kDa). Preferably component(i) is a polysaccharide, and more preferably it is dextran. As regardsits size, component (i) (which preferably is dextran) preferably has amolecular weight of at least 50 kDa, more preferably at least 70 kDa,yet more preferably at least 100 kDa or at least 200, 300 or even 500kDa. There is no specific upper limit for the molecular weight, althoughtypically it 1000 kDa or less, more typically 750 kDa or less, yet moretypically 500 kDa or less. A particularly preferred dextran is dextran500. A molecular weight within these preferred ranges (at least 50 kDa,etc) is particularly preferred in the context of the priming method ofthe invention.

As regards component (ii) for use in accordance with the methods andcompositions of the present invention, this may be a mixture of two orall of DMSO, DMG and valine, but typically just one of these agents isused. The agent valine is preferably L-valine. Generally, DMSO andvaline (typically L-valine) are more preferred, with DMSO being mostpreferred.

Preferably, component (i) is dextran and component (ii) is DMSO. Morepreferably, component (i) is dextran 500 and component (ii) is DMSO.

Component (i) is preferably brought into contact with the sample or cellfraction in the form of a composition, typically an aqueous solution.Preferably the composition is a saline solution. Preferably the pH ofthe solution is 6.8 to 7.8, e.g. at or around 7.4. Typically thesolution is buffered to the desired pH using a pharmaceuticallyacceptable buffer. Possible solvents for use in this regard include PBS(phosphate buffered saline), MOPS and HEPES, with PBS being preferred.

Component (ii) is preferably brought into contact with the sample orcell fraction in the form of a composition, typically an aqueoussolution. Preferably the composition is a saline solution. Preferablythe pH of the solution is 6.8 to 7.8, e.g. at or around 7.4. Typicallythe solution is buffered the desired pH. Possible solvents for use inthis regard include PBS (phosphate buffered saline), MOPS and HEPES,with PBS being preferred.

Preferably, components (i) and (ii) are combined into a singlecomposition before being brought into contact with the bloodcell-containing sample or cell fraction. This single compositiontypically consists essentially of the solvent (preferably PBS) and eachof components (i) and (ii).

The present invention also provides a method for preparingnon-erythrocyte blood cells for cryopreservation, which method comprises(a) a separation or priming method of the invention as defined herein,and (b) adding a cryoprotectant to the thus obtained non-erythrocyteblood cells.

The present invention also provides a method for the cryopreservation ofnon-erythrocyte blood cells, which method comprises (a) a separation orpriming method of the invention as defined herein, (b) adding acryoprotectant to the thus obtained non-erythrocyte blood cells, and (c)cryopreserving the non-erythrocyte blood cells.

The present invention also provides a method for the cryopreservationand subsequent recovery of non-erythrocyte blood cells, which methodcomprises (a) a separation or priming method of the invention as definedherein, (b) adding a cryoprotectant to the thus obtained non-erythrocyteblood cells, (c) cryopreserving the non-erythrocyte blood cells, and (d)thawing the non-erythrocyte blood cells. There may also be a furtherstep (e) of recovering the non-erythrocyte blood cells from the (by nowthawed) cryopreserved formulation.

Preferred formulations for the cryoprotectant are discussed above.Preferably the cryoprotectant comprises DMSO. In one embodiment it mayconsist essentially of DMSO.

Cryopreservation typically involves reducing the temperature to sub-zerotemperatures, i.e. less than 0° C. Typically it may involve thereduction of the temperature to −50° C. or less, such as to around −80°C. (e.g. −78° C.), but it may involve the reduction of the temperatureto −100° C. or less, such as to around −120° C. or less, or even −140°C. or less.

The present invention also provides a fraction of non-erythrocyte cellsobtainable by a method of the invention as defined herein. Typicallysuch cell fractions may be distinguished from cell fractions obtainedusing other methods by the characteristic high proportions of viablenon-erythrocyte blood cells. They may also be distinguished from cellfractions obtained via other methods by virtue of the protective effectthat will arise during subsequent cryopreservation step(s). There mayalso be a small remaining level of component (i) and/or (ii) in a cellfraction that has been prepared via a method of the present invention.

The present invention also provides a composition comprising (i) amacromolecular erythrocyte sedimentation enhancer, and (ii) DMSO, DMGand/or valine, which composition is suitable for use in recoveringnon-erythrocyte blood cells from a blood cell-containing sample and/orpriming non-erythrocyte blood cells to protect their integrity insubsequent cryopreservation and thawing steps, wherein if component (ii)is DMSO, then the concentration of DMSO in the composition is 10% v/v orless. Preferably in this regard, the concentration of component (ii)does not exceed 8% v/v, more preferably it does not exceed 6% v/v, yetmore preferably it does not exceed 4% v/v, and typically it does notexceed 3% v/v.

The concentration of component (i) in the composition may advantageouslybe 10% w/v or less, preferably 8% w/v or less, more preferably 6% w/v orless, yet more preferably 4% w/v or less, typically 3% w/v or less. Theconcentration of component (i) in the composition is preferably 0.1% w/vor more, more preferably 0.5% w/v or more, more preferably still 1% w/vor more, typically 2% w/v or more.

The concentration of component (ii) in the composition is preferably 8%v/v or less, more preferably 6% v/v or less, more preferably still 4%v/v or less, typically 3% v/v or less. The concentration of component(i) in the composition is preferably 0.1% v/v or more, more preferably0.5% v/v or more, more preferably still 1% v/v or more, typically 2% v/vor more.

In one preferred aspect of the composition, the concentration ofcomponent (i) is 1-5% w/v and the concentration of component (ii) is1-5% v/v.

The present invention also provides an apparatus comprising acomposition (comprising components (i) and (ii)) as defined herein,which apparatus is a bottle, a blood bag, a pre-filled syringe forinjection into a blood bag, or a kit comprising the composition, and ablood collection vessel.

The component (i) (e.g. dextran) and component (ii) (the dimethylcontaining component) together provide a surprisingly andsynergistically effective separation and/or priming medium which allowsfor (i) the enhanced partitioning into red and white cell fractions (andplasma fraction) of a sample comprising blood cells, (ii) improvedlevels of cell recovery following separation, and/or (iii) a protectiveeffect to be imparted to non-erythrocyte blood cells which protectstheir integrity in subsequent cryopreservation step(s). The separationmedium facilitates gravity assisted sedimentation and therefore step (b)generally requires nothing more than leaving the combination of sampleand separation medium to stand. Thus, typically no active steps areneeded in step (b), and it is possible simply to rely on gravity to helpbring about the desired sedimentation. For the avoidance of doubt,though, step (b) does not exclude the possibility of active steps beingtaken, provided that any such steps do not prevent the partitioning.

Step a) in the method of the invention for separating cells willtypically comprise a mixing step. A separation medium (i.e. acomposition comprising components (i) and (ii)) and bloodcell-containing sample are conveniently mixed at a ratio of 2: 1 to 1:2,preferably at or about a ratio of 1:1.

In a further aspect, the invention provides a composition suitable foruse as a blood cell separation medium comprising component (i) (e.g.dextran) and dimethylsulphoxide, dimethylglycine or valine. The use ofsuch a composition in a blood cell separation method constitutes afurther aspect of the present invention. In one aspect of the invention,the composition typically comprises 3-15, preferably 4-10% w/v dextranand 1-10, preferably 3-8% v/v dimethylsulphoxide, dimethylglycine orvaline.

The invention provides efficient and cost effective methods andcompositions for separating and recovering therapeutically ordiagnostically valuable cells from peripheral blood, umbilical cordblood, and bone marrow (e.g. a bone marrow aspirate); umbilical cordblood is especially preferred for treatment in accordance with themethods of the invention. In particular, the invention provides methodsand compositions for specifically removing the erythrocyte component ofthe blood.

The disclosed compositions and methods can be used, for example, toprepare cells for tissue culture, immunophenotypic characterization,other diagnostic testing, further purification and therapeuticadministration. In particular, the methods can be used to prepare cellsfor long term storage, e.g. cryopreservation. There is demand forstorage of cord blood when a child is born for the therapeutic potentialof cells within cord blood to address conditions later in life. Forexample, HSCs could be used to treat leukaemia and lymphoma; MSCs couldtreat heart disease, Alzheimer's Disease or Parkinson's Disease and beused to regenerate bone, cartilage, muscle or connective tissue; VSELscould regenerate neural, cardiac or muscle cells.

The therapeutic potential of pluripotent cells is increasing all thetime and the benefits of an autologous supply clear. However, there is acost associated with long term storage which means that enrichment forcells of interest (i.e. exclusion of erythrocytes to reduce the samplesize) is desirable. However, it is important that the small numbers ofkey cells, such as VSELs, are not further diminished by such separation.The present invention addresses these needs.

Cells can be recovered from either or both the sedimented phase(aggregate) or supernatant phase. According to the methods of theinvention, even very rare cell types can be recovered in relatively highyield. The sedimented phase contains the majority of erythrocytes fromthe sample while the supernatant contains the nucleated cells. Thenucleated cells are also referred to as the “white blood cells” andinclude the lymphocytes and stem cells. The “non-erythrocyte” cellsmentioned herein may preferably refer to one or more of these cells.

The disclosed compositions and methods can be used to isolate and enrichfor a variety of cell types, including, for example, T lymphocytes, Thelper cells, T suppressor cells, T killer cells, B cells, NK cells,hematopoietic stem cells, non-hematopoietic stem cells, VSELs or othercells in the blood circulatory system. The “non-erythrocyte” cellsmentioned herein may preferably refer to one or more of these cells.

The disclosed methods can be applied to cells of any mammalian bloodsystem including humans, non-human primates, rodents, swine, bovines,dogs, cats and equines. Samples containing human blood cells areespecially preferred. The blood cell-containing sample may be wholeblood or tissue or partially purified, e.g. to form a crude reduction inthe erythrocyte content. For the avoidance of doubt, the methods of thepresent invention are for use in connection with in vitro processing ofblood cell-containing samples, non-erythrocyte cells and cell fractions,i.e. the cells have been removed from the human (or animal) prior tocarrying out the methods of the invention.

According to the separation method of the invention, step (b), thepartition step, preferably lasts for at least 10 minutes, morepreferably at least 15 minutes, and can continue for e.g. up to 1 or 2(or more) hours, but typically no more than 1 hour is needed. Thus, thepartition step is typically 10 to 60 minutes, preferably 15-35 minutes.Preferably adequate partition is achieved within 30 minutes. ‘Adequatepartition’ can be measured as at least 90 or 95% of the maximumpartition the system can achieve if left for 12 hours.

According to the priming methods of the invention, the priming treatmenttypically involves contacting the cell fraction with components (i) and(ii) for at least 10 minutes, preferably at least 20 minutes. Primingcan continue for e.g. up to 1 or 2 (or more) hours, but typically nomore than 1 hour is needed, e.g. around 30 to 50 minutes is preferred.

Dextran is a polysaccharide consisting of glucose units linkedpredominantly in alpha (1 to 6) form. Dextran can cause stacking oferythrocytes (i.e., rouleau formation) and thereby facilitate theremoval of erythroid cells from solution. Typically, the concentrationof component (i) (e.g. dextran) in the cell separation composition is 10to 40 g/L (e.g., around 20 g/L). Occasionally the concentration ofcomponent (i) (e.g. dextran) used is higher than 20 g/L (e.g. 25 g/L oreven higher). Cell aggregation by rouleau can take an extended period oftime such that use of component (i) (e.g. dextran) alone is not acommercially viable separation technique.

In one aspect of the invention, the final concentration of component (i)(e.g. dextran) when mixed with the sample or cell fraction is typically1-20%, preferably 1.5 to 10%, more preferably 2.5-10% w/v.

References to dextran include dextran salts, e.g. dextran sulphatesodium salt. Dextran products vary significantly in their molecularweight; preferably, the dextran used in the present invention has amolecular weight of greater than 50, more preferably 50-1,000kilodaltons, especially preferred are molecular weights of 200-750, e.g.about 500 kilodaltons.

When DMSO, DMG or valine is combined with component (i) (e.g. dextran)and contacted with the blood cell containing sample, a greaterproportion of white blood cells can be recovered than if component (i)is used alone. Also, the thus obtained cells acquire some form ofprotection which helps preserve their integrity if the cells aresubsequently subjected to cryopreservation. Further, sedimentation of apopulation of erythrocytes, e.g. using TotiCyte 1×, may occur much morequickly than with dextran alone, preferably within 15-30 minutes.

In one embodiment, DMSO, DMG or valine is typically present at a finalconcentration (when mixed with the sample) of 0.25 to 10% w/v,preferably 0.5-5% w/v, more preferably 2-3%, e.g. about 2.5%.

In one embodiment, the ratio of component (i) (e.g. dextran) tocomponent (ii) (the dimethyl compound) is typically 1:1 to 1:5, moretypically 1:1 to 1:2, with a ratio of about 1:1 being preferred.

The cell separation composition of the invention typically comprises inan appropriate buffer (e.g. Phosphate buffered saline) and may containdivalent cations (e.g., Ca⁺² and Mg⁺²). Divalent cations can beprovided, for example, by a balanced salt solution (e.g., Hank'sbalanced salt solution), these are co-factors for selectin-mediated andintegrin-mediated cell-to-cell adherence.

One of the advantages of the present invention over some of the priorart methods is that it enables the separation/removal of erythrocytesfrom a blood cell-containing sample with excellent recovery levels forthe desired white blood cell fraction, without the need for antibodies.Thus, in one preferred aspect, the separation method of the presentinvention do not include the use of any antibodies, e.g. thecomposition(s) comprising components (i) and (ii) do not contain anyantibodies. This may (optionally) be true also for the compositions usedin the priming method of the invention. Notwithstanding this, as apractical matter it can be possible to introduce an antibody withoutprejudicing the benefits of the invention. Thus, the separationcomposition or medium of the invention may, but typically will not,additionally contain an appropriate antibody, antibody fragment, atandem antibody or another molecule capable of specifically recognisinga protein sequence or conformation. The separation compositions caninclude antibodies or fragments thereof to appropriate cell surfaceantigens, e.g. in order to encourage their co-sedimentation with theerythrocytes. The included antibodies or antibody fragments can beconjugated to a variety of functional groups known in the art (e.g.Streptavidin) which can be utilised to encourage sedimentation of atarget population of cells upon introduction of a binding partner (e.g.Biotin).

After a sedimentation step, unsedimented cells can be recovered from thesolution phase (i.e., the supernatant) by a variety of means, such ascentrifugation of the resulting supernatant fraction. Cells also can berecovered from the sedimented phase. Sedimented cells can be dissociatedby, for example, transferring the cells into buffers that containdivalent cation chelators such as EDTA or EGTA.

Cells recovered from the sedimented or unsedimented fraction can befurther separated, isolated or purified, e.g. by using antibodiesagainst cell surface antigens.

The separation method of the invention can be used to separate cellsfrom a variety of blood-cell containing samples, including peripheralblood (e.g., obtained by venipuncture), umbilical cord blood (e.g.,obtained post-gravida), and bone marrow (e.g., from aspirate).

Umbilical cord blood is preferably subjected to the method of theinvention within a week from birth, typically within 48, preferablywithin 24, more preferably within 12 hours. In a standard procedure, theumbilical cord is cleaned and blood bag needle inserted into theumbilical vein and arteries so that the blood passes into the bag whichis then clamped and sealed. A courier service may be used to transportthe sample to the site of performance of the cell separation method orit may be performed on the site of delivery of the infant.

The cell populations which are generated by the cell separation methodof the invention can be used in the context of allogeneic and autologoustransplantation. In the context of allogeneic transplantation, Tlymphocytes can be removed from the cell transplant to reduce Tlymphocyte-associated GvHD. In the context of autologoustransplantation, undesired cells such as metastatic cancer cells from apatient's blood or bone marrow can first be removed beforetransplantation. Desirable cells (e.g., hematopoietic stem cells) thencan be returned to the patient without, or substantially free of tumourcells.

The cell populations recovered according to the method of the invention,in particular the nucleated cells, preferably contain a good proportionof viable cells. The Examples describe, inter alia, a method ofassessing cell viability and, as an alternative, the Guava Personal CellAnalyser (PCA) Base System may be used to measure viability of a cellpopulation of interest. The proportion of viable cells as measured usingthese techniques is preferably greater than 50%, more preferably greaterthan 55 or 60%.

The separation method of the present invention typically serves,primarily, to exclude erythrocytes and retain nucleated cells within afraction of the starting sample and, as such, can be seen as a method ofpartial cell purification. However, the resulting fraction is likely tocontain MSCs, HSCs and VESLs and so subsequent processing steps ormodifications to the basic method described herein may be required toisolate a target population of interest. If a sample is cryopreserved,such subsequent steps may be performed when the sample is retrieved froma cryopreserved state.

The components of a composition for use according to the invention, i.e.a composition comprising components (i) and (ii) (also referred toherein as a cell separation composition) can be packaged individually orin admixture with one another. In a further aspect the present inventionprovides a kit comprising a cell separation composition of the invention(which may be in admixture or in separate containers) and a bloodcollection vessel (e.g., blood bag or vacuum tube). In some embodiments,the cell separation composition can be housed within a sterile bag.Furthermore, the sterile bag can be operably connected (e.g., viasterile tubing) to a processing bag, and the processing bag can beoperably connected (e.g., via sterile tubing) to a storage bag orstorage vessel to facilitate processing and sterile transfer of isolatedcells. A single sterile bag can also perform the role of the processingbag. The storage bag or vessel can include additional cryopreservativesuch as dimethylsulphoxide sufficient to provide a final concentrationof cryopreservative (DMSO) typically 1 to 10% of the final volume ofsolution. Cryopreservation can allow for long-term storage of thesecells for therapeutic or research use. The packaging material includedin a kit typically contains instructions or a label describing how thecell separation composition can be used to encourage the partitioning ofparticular types of cells. The kit may also contain a needle forcollection of blood, the needle being integral with or connected to theblood collection vessel.

In other embodiments, a single bag may be used for cell separation andcryopreservation, with e.g. DMSO providing both separation andcryopreservation functions (DMSO may be present at a final concentrationof around 2%). After the separation step within the bag, erythrocytesmay be expelled using a valve or tube at the bottom of the bag beforecryopreservation. The bag may be designed so that it can be hung duringthe separation step with the opening of the bag facing down so that onlyone opening is necessary for the filling of the bag and the removal ofthe erythrocytes.

Preferred features of the separation composition/medium discussedtherein in the Examples and generally in relation to the methods arepreferred features of the composition/medium per se and of the kitcomprising the composition/medium.

The invention is further described in the following non-limitingExamples and with reference to the figures in which:

FIG. 1 shows clear separation of the erythrocytes (bottom of test tube)from the nucleated cells (top of test tube) in blood samples that wereexposed to dextran 500 at a final concentration of 2.5% w/v and DMSO ata final concentration of either 2% v/v (left image) or 2.5% v/v (rightimage) for 30 minutes at room temperature.

FIGS. 2a and 2b show clear separation of the erythrocytes from thenucleated cells in blood samples that were exposed to dextran 500 at afinal concentration of 2.5% w/v and DMSO at a final concentrationvarying from 1% v/v to 5% v/v. From left to right, the following finalconcentrations of DMSO were analysed (% v/v): 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5 and 5. The samples were exposed to the above solutions for either 15minutes at room temperature (FIG. 2a ) or for 30 minutes at roomtemperature (FIG. 2b ).

FIG. 3 shows the erythrocyte volume fraction (haematocrit) of thenucleated cell fraction after cell separation. Blood samples were mixedat a ratio of 1:1 with either (i) PBS only (control, left), (ii) asolution containing 500 Mw dextran at a concentration of 5% w/v in PBS(final concentration of dextran of 2.5% w/v) (middle) or (iii) asolution containing 500 Mw dextran at a concentration of 5% w/v and DMSOat a concentration of 5% v/v in PBS (final concentration of dextran of2.5% w/v and of DMSO of 2.5% v/v) (right). Samples were left to separatefor 30 minutes at room temperature. Nucleated cell fractions (100 μl)were then transferred to a micropipette and centrifuged for 2 minutes at1500 rpm.

FIG. 4 shows a dot plot of 7-Aminoactinomycin D (7-AAD) fluorescencelevels (x-axis) against side scatter (y-axis). A blood sample was mixedat a ratio of 1:1 with a solution containing 500 Mw dextran at aconcentration of 5% w/v and DMSO at a concentration of 5% v/v in PBS(final concentration of dextran of 2.5% w/v and of DMSO of 2.5% v/v).Samples were then left to separate for 30 minutes at room temperaturebefore analysis was carried out using a “Stem Cell Enumeration Kit”obtained from Becton, Dickinson and Company.

FIG. 5 shows the separation of whole blood using constant 2.5% w/vDextran 70 and other substances. Row 1=β-alanine; row 2=L-proline; row3=L-valine; row 4=DMSO; and row 5=DMG. The left hand column shows eachof the separations at 15 minutes, with the right column showing it after30 minutes.

FIG. 6 shows a comparison of the separations of different Dextrans at aconstant concentration of DMSO, after 30 minutes (see Example 5). Topleft=Dextran 6 with control; Top right=Dextran 40; Bottom left=Dextran70; Bottom right=Dextran 500.

FIG. 7 shows a comparison of the separations of Dextran 500 v Dextransulphate sodium salt after 30 minutes.

FIG. 8 shows close ups of Dextran 500 and Dextran 500 sulphate sodiumsalt separations. These close ups were taken at the 30 minute mark todemonstrate the extra cloudiness of the Dextran sulphate sodium saltseparation.

FIG. 9 shows haematocrit levels after whole blood separation andcentrifugation. Prior to transferral to micropipettes, each mixture wasleft for 30 minutes to separate: the control had blood mixed 1:1 withPBS (left), the next tube had half Dextran half PBS mixed with blood(middle), the other had TotiCyte (right). Each were then transferred tomicropipettes and centrifuged.

FIG. 10 shows (on the left) separation after 5 minutes using Dextran 500at concentrations of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5% w/v (left toright) mixed with 2.5% v/v DMSO. On the right is the same but withDextran 70. FIGS. 11 and 12 show separation after 15 and 30 minutes,respectively.

FIG. 13 shows separation using a 1:1 by volume combination of Dextran500 at 2.5% w/v and DMSO at concentrations 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5, and 5% v/v. The left hand picture shows the separation after 15minutes, at which point the separation is good. The right hand pictureshows it after 30 minutes, where all concentrations work well and allhave produced clear white cell fractions.

FIG. 14 shows separation using a 1:1 by volume combination of Dextran 70at 1.5% w/v and DMSO at concentrations 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,and 5% v/v. The top two images show the Dextran at a constant 1.5%, onthe left at 15 minutes and on the right at 30 minutes. As can be clearlyseen, it did not really separate the blood faster than the blood willseparate out on its own. The bottom two images are of correspondingsamples where a 3% w/v solution of Dextran 70 was used.

FIG. 15 shows comparative separation tests using (1) solvent (PBS) only(as control), (2) DMSO (i.e. no component (ii)), (3) DMSO+HES, and (4)DMSO+dextran 500.

EXAMPLES Terms and Equipment Used in the Examples

“TotiCyte” refers to a combination of Dextran 500 and DMSO.

“TotiCyte 1×” refers to a TotiCyte composition based on PBS andcontaining 2.5% w/v Dextran 500 and 2.5% v/v DMSO.

“TotiCyte 5×” refers to a TotiCyte composition based on PBS andcontaining 7.5% w/v Dextran 500 and 7.5% v/v DMSO.

All experiments were performed using blood that had been mixed with CPDAas an anticoagulant unless otherwise stated. Also, unless statedotherwise, compositions added to blood cell-containing samples werebased on phosphate-buffered saline (PBS).

An FACS (Fluorescence-activated cell sorting) machine (FACSCalibur 4CA,serial number E4378) was used to measure the amount of CD34 and/or CD45cells in white blood cell fractions.

A Guave easyCyte 5 machine was used to measure the amount of TNC (totalnucleated cell count) in white blood cell fractions.

As regards methods for cell separation, three different methods wereadopted, namely Macopress, Syngen and manual. These methods were carriedout as follows.

Macopress

-   1. Turn on Macopress. It is important to do this before step 11, as    the Macopress cannot calibrate properly if a blood bag is already    hanging on the machine.-   2. Calculate how much blood+CPDA is in the blood bag.-   3. Sterile dock the blood bag to the Macopress processing kit (line    with white spike).-   4. Cut off the clamp nearest to the buffy coat bag (the one with two    tubes coming out of it, as opposed to one like the plasma bag), and    close all other clamps.-   5. Using the port on the collection bag line, add in an equal volume    of 1× TotiCyte.-   6. Agitate the bag both whilst and after completion of adding    TotiCyte.-   7. Use the syringe previously used to add the TotiCyte to remove as    much air from the bag as possible (the Macopress buffy coat bag only    holds 200 ml so make sure not to allow it to fill up with air before    it can finish pressing).-   8. Hang up the Macopress processing bag onto the Macopress and allow    to separate for 30 minutes.-   9. Using the arrows, choose program 4.-   10. Follow the instructions given on the screen—insert the tubing    into the clamps that are lit up (1, 3 and OPTI). Ensure there are no    kinks in the line. Break the breakventile on the top of the bag and    ensure the clamps on the buffy coat line are open, and those on    other parts of the processing set are closed.-   11. Press Enter to start the Macopress.-   12. Press Escape once the Macopress has finished, to ensure that the    machine does not seal the line at clamp 1.-   13. Remove the bag and tubing from the Macopress.-   14. Pull apart the seal between the two bags. Cut the tube of the    buffy coat bag to allow the contents of the line to drain in. Heat    seal this tube before proceeding to the next step.-   15. Place the set snugly into a centrifuge bucket, aiming to ensure    that the buffy coat bag will not crease up during the centrifugation    step, and ensure that all clamps are closed.-   16. Ensure the centrifuge is properly balanced, and then centrifuge    at 500 g for 15 minutes.-   17. Carefully remove the processing set from the centrifuge so as    not to disturb the pellet.-   18. Place the bag back onto the Macopress, this time putting the    tubing into clamps 1 and 3 only.-   19. Select program 3 (10 ml final volume), break the breakventile on    top of the buffy coat bag and ensure all clamps on the extra tubing    are closed before pressing Enter.-   20. Remove the processing set from the Macopress and pull apart the    seal between the two bags. This will leave a plasma bag and a buffy    coat bag containing 10 ml.-   21. Rub the buffy coat bag gently to remove all of the pellet from    the ridges and then remove the buffy coat from the bag using a 20 ml    syringe.-   22. Measure exactly how much buffy coat there is (for working out    the recovery).-   23. TNC and viability can then be tested on the Guava easyCyte    machine, and CD34 and CD45 cells and their viabilities on the FACS    machine.

Syngen

-   1. Remove the blood from the Fenwal blood bag using a 50 ml syringe    (or more than one if necessary). Record the volume removed.-   2. Retain a small amount of this whole blood for use later on in    working out TNC recovery.-   3. Replace the blood back into the blood bag.-   4. Measure out an equal volume of 1× TotiCyte.-   5. Add this to the blood bag and mix well.-   6. Remove the pin from the bottom of the cartridge and the cap from    the blue filter.-   7. Sterile dock the blood bag to the Syngen cartridge's central    line.-   8. Allow the blood/TotiCyte mix to flow into the cartridge.-   9. Seal the line as close to the top of the cartridge as possible.-   10. Invert the cartridge to further mix the sample, making sure to    avoid getting any liquid into the 2 tubes at the side of the    cartridge (tubes that go through to the other chambers) or into the    blue filter.-   11. Leave the sample to separate for 30 minutes.-   12. Attach the cartridge to the Syngen processing module and switch    it on.-   13. Balance the centrifuge by using a Syngen cartridge filled with    water attached to a processing module.-   14. Spin the cartridge using program 1—this will go through 4 spin    cycles which are each a different length and speed.-   15. Remove the cartridge from the centrifuge, transferring the    cartridge to the Syngen workstation and the processing module to the    docking station (the module will now be displaying a P, indicating    that the data needs to be processed).-   16. Download the information from the module, entering in the    information requested (centrifuge number, sample name, cartridge lot    number and processing module lot number used).-   17. The buffy coat chamber should contain 20 ml: remove this and    measure exactly how much there is, as it can vary slightly.-   18. Mix the buffy coat well then take a sample from it for viability    analysis.-   19. TNC and viability can then be tested on the Guava easyCyte    machine, and CD34 and CD45 cells and their viabilities on the FACS    machine.

Manual Method

-   1. Calculate the volume of blood+CPDA.-   2. Retain a small amount of this whole blood for use later on in    working out TNC recovery.-   3. Transfer this into a separation funnel attached to a retort    stand. Ensure that the tap at the bottom is closed.-   4. Add in an equal volume of 1× TotiCyte. Remove the lid to get rid    of any excess air, and leave to separate for 30 minutes.-   5. Place a falcon tube or dish underneath the funnel. Turn the tap    to open the nozzle only slightly and allow the red fraction to flow    out of the funnel.-   6. Keep a close eye on the amount of red left: do not remove all of    it as the interface will be lost if absolutely all of the red is    removed.-   7. Once sufficiently close to the interface, turn off the tap. Allow    as much of the red to drip from the nozzle before removing the tube    from underneath and replacing it with a new one.-   8. Open the tap again to allow the white cell fraction to be    aspirated.-   9. Spin down the white cell fraction for 10 minutes at 500 g and    re-suspend in 10 ml of supernatant.-   10. TNC and viability can then be tested on the Guava easyCyte    machine, and CD34 and CD45 cells and their viabilities on the FACS    machine.

Example 1 General Cell Separation Method

Umbilical cord blood was collected into standard Baxter 250 ml (nominal)blood bags containing 35 ml of a citrate phosphate dextrose adenine(CPDA) anticoagulant solution.

A solution containing dextran and a further constituent selected fromdimethyl sulphoxide (DMSO), dimethyl glycine (DMG), L-valine, L-proline,β-alanine, leucine, isoleucine and glycine, in phosphate-buffered saline(PBS), was prepared. This solution was added to a blood sample at aratio of 1:1, this was then mixed thoroughly. Separation of theerythrocyte and nucleated cell fractions took place at room temperaturewithin a time period of 15 or 30 minutes.

Separation of the cell fractions could be observed visually. FIG. 1shows clear separation of the erythrocytes (bottom of test tube) fromthe nucleated cells (top of test tube). This image was taken 30 minutesafter exposure to dextran (500 molecular weight (Mw)) at a finalconcentration of 2.5% w/v and DMSO at a final concentration of either 2%v/v (left image) or 2.5% v/v (right image).

Similar cell separation as described above was seen when dextran 500 Mwwas replaced with dextran at a lower molecular weight of 70 Mw at afinal concentration of between 3% and 5% w/v (data not shown). Similarcell separation was also seen when dextran 500 Mw was replaced withdextran 500 sulphate sodium at a final concentration of between 2.5% and5% w/v (data not shown).

Blood samples that were exposed to dextran 70 Mw at a finalconcentration of 2.5% and DMG or L-valine at a final concentration of2.5% w/v also showed a clear separation of erythrocytes and nucleatedcells after 30 minutes (data not shown). By contrast, blood samples thatwere exposed to dextran 70 Mw at a final concentration of 2.5% w/v andL-proline, β-alanine, leucine, isoleucine or glycine at a finalconcentration of 2.5% w/v showed little if any separation oferythrocytes and nucleated cells after 30 minutes (data not shown here).

It was also observed that blood samples exposed to dextran 500 Mw at afinal concentration of 2.5% w/v and DMSO at final concentration of 1% to5% v/v showed a clear separation of erythrocytes and nucleated cellsafter 15 minutes and 30 minutes (FIGS. 2a and 2b ).

For the above experiments, substantial separation was often seen after15 minutes of exposure, although further separation would be seen afterbetween 15 minutes and 30 minutes of exposure. This is noticeable, forexample, when the samples of FIG. 2a are compared against the samples ofFIG. 2 b.

Example 2 Erythrocyte Volume Fraction (Haematocrit) Study

In this Example, the levels of erythrocyte volume fraction remaining inthe nucleated cell fraction after cell separation were determined. Threetubes were prepared for the separation: (i) blood sample mixed at aratio of 1:1 with PBS only (control), (ii) blood sample mixed at a ratioof 1:1 with a solution containing 500 Mw dextran at a concentration of5% w/v in PBS (final concentration of dextran of 2.5% w/v), (iii) bloodsample mixed at a ratio of 1:1 with a solution containing 500 Mw dextranat a concentration of 5% w/v and DMSO at a concentration of 5% v/v inPBS (final concentration of dextran of 2.5% w/v and of DMSO of 2.5%v/v). Samples were left to separate for 30 minutes at room temperature.

It was observed that the cell fractions in sample (iii) separated at afaster rate than in sample (ii) and, after 30 minutes, sample (iii) haddeveloped a more compact erythrocyte fraction compared to sample (ii)(data not shown).

Nucleated cell fractions (100 μl) were transferred to a micropipette,and the ends were closed using plasticine. Micropipettes were thencentrifuged for 2 minutes at 1500 rpm.

FIG. 3 shows the micropipettes after centrifugation. The control (leftpipette in FIG. 3) shows a high packed erythrocyte volume within thewhite cell fraction. By contrast, samples (ii) (middle pipette) and(iii) (right pipette) showed a much reduced packed erythrocyte volume ofapproximately 1%.

Example 3 Assessment of Cell Viability and the Presence of HematopoieticStem Cells (HSCs) Using Flow Cytometry

A blood sample was mixed at a ratio of 1:1 with a solution containing500 Mw dextran at a concentration of 5% w/v and DMSO at a concentrationof 5% v/v in PBS (final concentration of dextran of 2.5% w/v and of DMSOof 2.5% v/v). Samples were left to separate for 30 minutes at roomtemperature.

A sample of the resulting nucleated cell fraction was then analysedusing flow cytometry. In particular, analysis was carried out using a“Stem Cell Enumeration Kit” obtained from Becton, Dickinson and Company.Analysis was carried out as per the Application Guide provided with theKit and the templates used are based on a method featured in theClinical and Laboratory Standards Institute H42-A2 approved guideline(Enumeration of Immunologically Defined Cell Populations by FlowCytometry; Approved Guideline-Second Edition. Wayne, Pa.: Clinical andLaboratory Standards Institute; 2007. CLSI document H42-A2).

FIG. 4 shows a dot plot of 7-Aminoactinomycin D (7-AAD) fluorescencelevels (x-axis) against side scatter (y-axis). 7-AAD is a fluorescentcompound used to assess cell viability. It permeates the cell membranesof non-viable cells only, and so a low level of fluorescence (asrepresented by the rectangular gate on the dot plot) represents thecells that are viable. This dot plot shows that the majority of thetotal cell population are viable.

The flow cytometric analysis also showed that the cell separation methoddescribed above is an effective way of isolating a HSC population from ablood sample (data not shown).

Example 4 Dextran 70 with Either DMSO or an Amino Acid

Whole blood samples were mixed with 1:1 with PBS compositions comprising2.5% Dextran 70 plus DMSO, L-valine, L-proline, β-alanine, leucine,isoleucine, glycine and DMG. Each of the amino acids and the DMSO weretested at concentration levels of 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%and 5% in the PBS composition. After mixing, each sample was left for 30minutes at room temperature. FIG. 5 shows images of the samples after 15minutes and also after 30 minutes.

β-alanine did very little, even after 30 minutes. L-proline did verylittle after 30 minutes also. The tube with a 1% concentration separatedout at a similar rate to the 2% tube in the L-valine experiment. In thisregard, it may be worth noting that the results for some of the higherconcentration amino acid solutions could have been affected bylimitations in the solubility of the amino acid. L-valine performedbetter than DMG: the white cell fraction after 30 minutes was muchclearer with the L-valine. None of isoleucine, leucine and glycineseemed to induce any sedimentation at all. FIG. 5 shows each of thesubstances described at the separation, except glycine, leucine andisoleucine are not shown due to their almost total inactivity—all ofthem separated out the blood no faster than blood mixed 1:1 with PBS.Below is a list of the order from best to worst of each substance at theseparation:

DMSO>L-valine>DMG>L-proline>β-alanine>Leucine/isoleucine/glycine

Example 5 Dextran Sizes and Types

Different molecular weights of Dextran were tested. In particular,Dextran 6, 40, 70 and 500 were compared. Stocks of 2-10% w/v (2%, 3%,4%, 5%, 6%, 7%, 8%, 9% and 10%) of each molecular weight of Dextran wereset up, to give concentrations of 1-5% once combined with the bloodsample on a 1:1 basis by volume. The stocks all contained the sameconcentration of DMSO, namely 2.5% v/v.

Dextran 6 and Dextran 40 didn't really separate out, either by 15minutes or the full 30 minutes, as can be seen in FIG. 6. Dextran 70 didstart to separate out quite well, with a clear gradient—the 5%concentration separated out much quicker and better than the lowerconcentrations. After 30 minutes, the Dextran 70 still had this cleargradient, with the higher concentrations separating over halfway downthe tube. The Dextran 500 was clearly the best at the separation. The 3%tube had its red cell fraction almost completely compacted by the 15minute mark, and after 30 minutes, all the tubes bar the 1%concentration were extremely well compacted: all of the tubes from 2.5%upwards looked essentially the same (some had slightly cloudier whitecell fractions).

FIG. 6 shows images of the samples after 30 minutes. The Dextran 6 and40 did not separate out any faster than the control (blood diluted withPBS). There is a clear gradient for the Dextran 70, but at lowerconcentrations it did not really separate out. The Dextran 500 separatedthe blood well and gave a clear white cell fraction.

Example 6 Dextran 500 v Dextran Sulphate Sodium Salt

Dextran 500 (at 2.5% w/v) was mixed with DMSO at 2.5% v/v; the same wasdone with the Dextran sulphate sodium salt. lml of each composition wasplaced in a tube, and each tube had lml of blood added, and was thenleft to separate for 30 minutes, as with most of the other experiments.

Dextran 500 sulphate sodium salt separated the blood out relativelywell, but not quite as well as the Dextran 500/DMSO combination, as seenin FIG. 7. The compactness was very similar, but the white cell fractionwas much cloudier in the Dextran salt, with many red cells visiblyfloating around just above the red cell fraction, as can be seen in FIG.4 below. Thus, while both are effective, the Dextran 500 provided aslightly clearer white fraction.

FIG. 8 shows close ups of Dextran 500 and Dextran 500 sulphate sodiumsalt separations resulting from following the protocol described abovein this Example. These close ups were taken at the 30 minute mark todemonstrate the extra cloudiness of the Dextran sulphate sodium saltseparation.

At this point it is worth noting that while all aspects of the inventionare believed to offer advantages over the prior art, the Dextran500/DMSO combination, especially when used in the form of a compositionobtainable by mixing equal volumes of (i) 2.5% w/v Dextran 500 in PBSwith (ii) 2.5% v/v DMSO in PBS), stands out from the experimentsdiscussed in this Example and preceding Examples as particularlyadvantageous.

Example 7 Haematocrit Experiment

The level of haematocrit remaining in the white cell fraction wasinvestigated. Three tubes were prepared for the separation, eachcontaining a 1:1 mixture of blood:PBS-based composition (the firstcomposition was PBS alone, as a control; the second composition had 5%w/v Dextran 500 mixed 1:1 with PBS (to give 2.5% w/v); the thirdcomposition was TotiCyte 1×) and left for 30 minutes at room temperatureto ensure maximum separation. The sample with TotiCyte 1× separatedslightly faster than the one with just Dextran. Both remained relativelycloudy until after 30 minutes of separation, at which point the TotiCytehad developed a more compact red cell fraction. 100 ul of the white cellfraction was transferred to a micropipette, and the ends closed up usingplasticine. The micropipettes were then centrifuged for 2 minutes at1500 rpm. Both the Dextran only and the TotiCyte samples had around 1%haematocrit and seemed much more compact than the control. To the nakedeye there were no differences in the amount of haematocrit in theDextran only and the TotiCyte tubes, as can be seen in FIG. 9.

In addition, samples were tested for haematocrit levels both using theMacopress machine and manually. The Macopress produced fairly consistentresults as the machine was set to only collect a certain level ofhaematocrit (the Macopress stops the press action when a given level ofred cells travel through the sensor, so in theory the amount that itleaves should always be the same). Values above 1% residual haematocritonly arose for runs where the sensitivity of the sensor had beenaltered. It was more difficult to get a constant level of haematocritwith the manual method, as to some extent, how much red to take offwithout taking the interface is subjective.

Example 8 Varying Concentrations of Dextran 70 and 500

With the aim of determining optimum concentrations of DMSO and Dextran,varying concentrations of Dextran 70 and 500 (1, 1.5, 2, 2.5, 3, 3.5, 4,4.5, and 5% w/v) were mixed with 2.5% v/v DMSO and blood (as usual,equal volumes of the Dextran and DMSO solutions were combined, and thena volume of blood added corresponding to that of the combined Dextranand DMSO solution), and left to separate for 30 minutes. The samplesderived from the Dextran 500 components with 2.5%-4.5% started toseparate out quite well even after 5 minutes. After 5 minutes, theDextran 70 appeared not to have changed. FIG. 10 shows images of thesamples after 5 minutes.

After 15 minutes, the majority of the separation had finished for eachof the samples with Dextran 500 (except 1%), although after 30 minutesthe fractions did become more compacted and clearer. The Dextran 70 hadnot done much after 15 minutes, although the tube at a concentration of5% Dextran 70 had started to make some headway on the compaction; FIG.11 shows images of the samples after 15 minutes. After 30 minutes,though, there was a very clear gradient: the first four tubes (1, 1.5, 2and 2.5%) had not really separated out, but the tubes at 3-5% hadseparated well, although were not as compact or have such clear whitecell fractions as the samples with corresponding concentrations ofDextran 500; FIG. 12 shows images of the samples after 30 minutes.

The second half of this experiment involved changing the concentrationof DMSO, whilst keeping the Dextran concentration constant. To this end,2.5% w/v Dextran 500 was mixed with varying concentrations of DMSOsolutions (1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5% v/v). For comparison,equivalent tests were also done using a 1.5% w/v Dextran 70 solution andalso a 3% w/v Dextran 70 solution (in place of the Dextran 500). After15 minutes, the Dextran 500/DMSO tubes had all compacted the red cellfraction down quite well—to about ⅓ of the tube. The 5% tube was theonly one not to be quite so compacted at this stage. However, after 30minutes all tubes had compacted even further and had much clearer whitecell fractions. The sample derived from the 1.5% Dextran 70 solution didnot appear to work any faster than blood mixed with PBS that had beenleft to separate (control). There was little/no change in the clarityand compactness of the Dextran 70 concentrations even after 30 minutes.FIGS. 13 and 14 show images of the samples. The bottom two images inFIG. 14 are of the sample derived from the 3% Dextran 70 solution. Thismade much more difference on the separation, and at 30 minutes, definedwhite and red cell fractions had developed. The red cell fraction wasnot as well compacted as it was with Dextran 500, but it was muchclearer than in the sample derived from the 1.5% Dextran 70 solution.

Example 9 Actual Numbers of Cells Recovered in the White Cell FractionsCD34 and CD45 Count

Separations were carried out on two different blood samples. For eachsample, a separation was done using (a) TotiCyte 1×, and (b) a 2.5% w/vDextran 500 solution. The 2.5% Dextran solution was mixed with lml ofblood such that the final concentration of Dextran in the sample was thesame as in the TotiCyte replicate (in which both the DMSO and Dextran500 components have a final concentration of 1.25% [v/v and w/v,respectively]). After 30 minutes or so at room temperature, once theseparation had progressed as far as it would go, the white cell fractionwas removed and transferred to another tube. 100 ul of this was run onthe FACS machine to test for CD34 and CD45 cells. The effect ofintroducing the DMSO (in the TotiCyte sample) on the quantity of cellsrecovered was as follows:

-   -   On average, separation using TotiCyte recovered 29% more CD34        cells than when the Dextran only solution was used    -   On average, separation using TotiCyte recovered 7% more CD45        cells than when the Dextran only solution was used

In terms of cell viability %, the proportion of the recovered cells thatwere viable was slightly lower when the TotiCyte composition was used ascompared to the Dextran only solution, but this was generally more thanoffset by a greater number of cells being recovered when the TotiCytecomposition was used (meaning that there were significantly more viablecells recovered using the TotiCyte composition).

Further Testing of TNC (Total Nucleated Cell) Count & C34 Count

Corresponding testing was also done to confirm the effectiveness of theTotiCyte 1× across a larger sample size. In addition, correspondingtesting was also done to confirm the viability of using TotiCyte 5×. Theresults are summarised in the following table.

Composition Processing used method TNC recovery % CD34 recovery %TotiCyte 1X Macopress 97*  79* Manual 99  87 Syngen 78* 90 Any of theabove 91*  81* TotiCyte 5X Macopress 66  68 *average values taken from 9samples in the case of the Macopress method and from 2 samples in thecase of the Syngen method

The testing showed that TotiCyte 1× enabled consistently high TNCrecovery rates, regardless of the choice of method (i.e. using a manualmethod, the Syngen system or the Macopress). CD34 cell recovery was alsohigh. The viability of using the more concentrated TotiCyte 5×composition was confirmed, although the Toticyte 1× compositiongenerally provided the highest recovery rates.

Post-Thaw Analysis

Further testing was done to compare the effects of the proposedseparation methods following downstream addition of DMSO to knowncryoprotective levels, freezing, and then thawing. In each case, theaddition of DMSO prior to freezing was done following the approachdescribed in Example 11 below, and the same freeze thaw process wasused. When DMSO was added prior to freezing, it was added at 4° C., andwas added slowly so as not to shock the cells; the samples were frozenin a validated passive freeze box with a fluid-filled blood bag on top(how the boxes were validated); samples were thawed quickly at 37° C. byswirling and gently inverting the tube, before being moved into wet iceas soon as the mixture became slushy; the blood was sampled quicklyafter complete thawing to ensure maximum retention of viability. Theresults are summarised in the table below, alongside correspondingresults for whole blood samples. For the washing step, thawed cellsuspension was centrifuged at 500 g for 10 min; the supernatant wasdiscarded and cell pellet gently resuspended in cold PBS; the cellsuspension was centrifuged again at 500 g for 10 min and the pelletresuspended in cold PBS to the volume desired.

Sample washed Composition used prior to Average TNC Average CD34 (samplesize) cell count? recovery % recovery % N/A - whole blood No 128 86 used(5) Toticyte 1X (5) 107 81 TotiCyte 5X (3) 79 91 N/A - whole blood Yes58 71 used (5) Toticyte 1X (5) 66 66 TotiCyte 5X (3) 34 27

The samples treated with TotiCyte 1× and TotiCyte 5× both provided goodpost thaw recovery levels, with the TotiCyte 1× proving particularlyeffective. There was a significant difference between the viabilities ofeach whole blood and its corresponding TotiCyte 1× and 5× replicates.For the most part, it was found during the course of these experimentsthat whole blood does not always survive the thawing processparticularly well. Thus, notwithstanding inevitable variations betweendifferent blood samples, it was found that while more cells weregenerally recovered from the whole blood cell samples, recovery figureswere generally comparable to the TotiCyte 1× replicate once viabilitywas taken into account. Indeed, the average TNC count (66%) for thesamples treated with TotiCyte 1× and subjected to washing was evenhigher than the corresponding average TNC count (58%) for whole blood.Similarly, the average CD34 count (91%) for the samples treated withTotiCyte 5× and not subjected to washing was even higher than thecorresponding CD34 count (86%) for whole blood. (It is worth noting inconnection with the above results that while recovery levels of >100%are of course not possible, levels >100% can nonetheless be recorded intesting, due e.g. to variation within blood samples.)

Example 10 Different Anticoagulants

A blood sample was processed using TotiCyte 1×, but with half containingCPD and the other half containing CPDA. Both were processed manually inseparation funnels, with the red cell fraction being siphoned off firstbefore collection of the white cell fraction. Both were tested on theFACS machine and Guava easyCyte for CD34/CD45 cells and TNC,respectively. The results are as follows.

Average TNC recovery % Average CD34 recovery % Anticoagulant (samplesize = 2) (sample size = 2) CPD 80 57 CPDA 79 81

The TNC recovery between the two anticoagulants is similar. Testing ofCD45 recovery % showed that 87% were recovered with the CPDA fraction,compared to 70% CD45 with the CPD fraction (data not shown). Bothanticoagulants are suitable for use according to the invention, thoughthere may be instances where CPDA is preferred.

Example 11 Priming a Cell Fraction for Freezing to Enhance Post ThawRecovery

The following method was followed to test the effect on post thaw cellrecovery of carrying out the priming method of the present inventionprior to contacting the cell fraction with a cryoprotectant and freezingthen thawing it.

-   1. Trypsinise was used to remove the cells from the flask.-   2. The cells were spun down to remove the trypsin.-   3. The cells were then resuspended in 10 ml DMEM.-   4. TNC and CD45/CD34 cell counts were measured (in order to    determine post thaw cell recovery).-   5. 0.5 ml was aliquoted out into each of eight 2 ml cryovials.-   6. A further 0.5 ml DMEM was added to the first two tubes, 0.5 ml of    TotiCyte 1× to each of the next two, 0.1 ml of TotiCyte 5× to each    of the next two, and 0.5 ml of 2.5% w/v Dextran 500 to the final two    tubes. The samples were then mixed well.-   7. The samples were then left for 45 minutes at room temperature.-   8. DMSO was added to one of each pair of tubes (i.e. one of each of    the four types) up to 7.5%. To the other four tubes, a combination    of DMSO and FCS (fetal calf serum) was added.-   9. The cell samples were transferred to a Mr Frosty freezing    receptacle.-   10. The bottom compartment was filled with methanol, and the whole    container placed in a −80° C. freezer for at least one night.-   11. The container was removed from the freezer and thawed quickly.-   12. Part of the fraction was removed to another tube for a retest    without washing.-   13. To the cells remaining after the removal of this small aliquot,    half the volume of warmed DMEM was added dropwise, with the tube    being swirled gently all the time.-   14. The samples were then centrifuged for 10 minutes at 500 g.-   15. The supernatant was removed and resuspended in 0.5 ml DMEM.-   16. The samples were then retested for cell recovery.

The above protocol was followed, including leaving the samples in thefreezer for 2/3 days before thawing. The TNC recovery (as measured onGuava PCA) results are set out below.

Pre-freeze Post-thaw Composition used Cryopreservant viability %viability % DMEM DMSO 90.2 77.67 DMEM DMSO/FCS 90.2 69.75 TotiCyte 1XDMSO 90.2 87.13 TotiCyte 1X DMSO/FCS 90.2 91.03 TotiCyte 5X DMSO 90.273.9 TotiCyte 5X DMSO/FCS 90.2 76.1 Dextran only DMSO 90.2 82.9 Dextranonly DMSO/FCS 90.2 81.6

Both Dextran and DMSO had a priming effect on cells, as the TotiCyte andDextran cell recoveries were both significantly better than the control.After a wash step, the TotiCyte 1× appeared to be the best atmaintaining TNC post-thaw, as well as retaining 85%+ viability, followedby the Dextran alone, then 5× TotiCyte.

Example 12 Further Comparative Testing

Further blood separations were carried out using the followingcompositions:

-   -   Blood+equal volume of PBS as control (test 1)    -   Blood+equal volume of PBS with 2.5% v/v DMSO, such that the        concentration of DMSO following mixing with the blood sample was        1.25% v/v (test 2)    -   Blood+equal volume of PBS with 2.5% w/v HES (hydroxylethyl        starch) and 2.5% v/v DMSO (test 3)    -   Blood+equal volume of TotiCyte 1×, i.e. PBS with 2.5% w/v        dextran 500 and 2.5% v/v DMSO (test 4).

Images of the separation achieved in these tests appear in FIG. 15. Asis evident, the DMSO-only composition used in test 2 performed no betterthan the control (test 1)—neither achieving any separation over thecourse of the test (60 minutes). Comparable separation was seen in tests3 and 4, confirming HES as a viable alternative to dextran.

1. A method for priming non-erythrocyte blood cells forcryopreservation, said method comprising contacting the cells with DMSO,DMG and/or valine, wherein when the cells are contacted with the DMSO,DMG and/or valine, the concentration of DMSO, DMG and/or valine does notexceed 5% v/v.
 2. A method according to claim 1, wherein thenon-erythrocyte blood cells are present in the form of a non-erythrocyteblood cell fraction that has already undergone a treatment(s) to removeerythrocytes.
 3. A method according to claim 1, wherein thenon-erythrocyte blood cells are present in the form of a bloodcell-containing sample which comprises said non-erythrocyte blood cells.4. A method according to claim 3, wherein the sample is whole blood. 5.A method according to claim 1, wherein the non-erythrocyte blood cellscomprise white blood cells.
 6. A method according to claim 1, whereinthe method is for increasing the proportion of viable white blood cellsrecovered following a subsequent cryopreservation.
 7. A method accordingto claim 1, wherein the non-erythrocyte blood cells are taken from amammal.
 8. A method according to claim 1, wherein the non-erythrocyteblood cells are taken from a human.
 9. A method according to claim 1,which comprises contacting the non-erythrocyte blood cells with acombination of: (i) a macromolecular erythrocyte sedimentation enhancer,and (ii) the DMSO, DMG and/or valine; wherein when the bloodcell-containing sample is contacted with components (i) and (ii), theconcentration of component (ii) does not exceed 5% v/v.
 10. A method forpriming a non-erythrocyte blood cell fraction for cryopreservation, saidmethod comprising contacting the cell fraction with a combination of:(i) a macromolecular erythrocyte sedimentation enhancer, and (ii) DMSO,DMG and/or valine; wherein when the blood cell-containing sample iscontacted with components (i) and (ii), the concentration of component(ii) does not exceed 5% v/v.
 11. A method according to claim 10,comprising contacting the cell fraction with a composition comprisingcomponents (i) and (ii), wherein the cell fraction and the compositionare mixed at a ratio of 10:1 to 1:10, preferably 5:1 to 1:5, morepreferably 2:1 to 1:2.
 12. A method according to claim 9, comprisingcontacting the non-erythrocyte blood cells with a composition comprisingcomponents (i) and (ii), wherein the composition comprises 0.1-10% w/vof component (i) and 0.1-10% v/v of component (ii).
 13. A methodaccording to claim 9, wherein, once the non-erythrocyte blood cells havebeen contacted with components (i) and (ii), the concentration ofcomponent (i) is 0.25 to 5% w/v, and the concentration of component (ii)is 0.25 to 5% v/v.
 14. A method according to claim 9, wherein component(i) is dextran and component (ii) is DMSO.
 15. A method according toclaim 9, wherein component (i) is dextran having a molecular weight ofat least 50 kDa.
 16. A method according to claim 9, wherein component(i) is dextran 500 and component (ii) is DMSO.
 17. A method forpreparing non-erythrocyte blood cells for cryopreservation, which methodcomprises (a) a method as defined in claim 1, and (b) adding acryoprotectant to the thus obtained non-erythrocyte blood cells.
 18. Amethod for the cryopreservation of non-erythrocyte blood cells, whichmethod comprises (a) a method as defined in claim 1, (b) adding acryoprotectant to the thus obtained non-erythrocyte blood cells, and (c)cryopreserving the non-erythrocyte blood cells.
 19. A method for thecryopreservation and subsequent recovery of non-erythrocyte blood cells,which method comprises (a) a method as defined in claim 1, (b) adding acryoprotectant to the thus obtained non-erythrocyte blood cells, (c)cryopreserving the non-erythrocyte blood cells, and (d) thawing thenon-erythrocyte blood cells.
 20. A method according to claim 17, whereinthe cryoprotectant comprises DMSO.
 21. A composition comprising (i) amacromolecular erythrocyte sedimentation enhancer, and (ii) DMSO, DMGand/or valine, which composition is suitable for use in recoveringnon-erythrocyte blood cells from a blood cell-containing sample and/orpriming non-erythrocyte blood cells to protect their integrity insubsequent cryopreservation and thawing steps, wherein if component (ii)is DMSO, then the concentration of DMSO in the composition is 10% v/v orless.
 22. A composition according to claim 21, wherein component (i) isdextran and component (ii) is DMSO.
 23. A composition according to claim21, wherein component (i) is dextran having a molecular weight of atleast 50 kDa.
 24. A composition according to claim 21, wherein component(i) is dextran 500 and component (ii) is DMSO.
 25. A compositionaccording to claim 21, wherein the concentration of component (i) is1-5% w/v and the concentration of component (ii) is 1-5% v/v.
 26. Anapparatus comprising a composition as defined in claim 21, whichapparatus is a bottle, a blood bag, a pre-filled syringe for injectioninto a blood bag, or a kit comprising a composition as defined in claim21 and a blood collection vessel.